Analyzing cross‐talk of EPO and EGF genes along with evaluating therapeutic potential of Cinnamomum verum in cigarette‐smoke‐induced lung pathophysiology in rat model

Abstract The integrity of the distal alveolar epithelium is crucial for lung regeneration following an injury. The present study aimed to evaluate the effect of Cinnamomum verum extract; cross‐talk of epidermal growth factor (EGF) and erythropoietin (EPO) genes in a smoke‐induced lung injury rat model. For experimentation (n = 27), albino rats were divided equally into three groups, i.e., negative control (NC), positive control (PC), and treatment group (TG). Cigarette smoke was exposed to PC and TG (4 CG/day). C. verum was given orally (350 mg/kg body weight) for 21 days. Decapitation (n = 3) was done on 14th, 18th, and 21st days, respectively. Analyses (hematology, biochemical, high performance liquid chromatography [HPLC], histology, and gene expression) were carried out and results were statistically analyzed by two‐way analysis of variance. HPLC analysis of ethanolic extract of C. verum was done to identify the presence of phenolic constituents which showed high concentrations of quercetin and P‐coumaric acid. Serum oxidative parameters such as total oxidant status, malondialdehyde, and hematological parameters such as red blood cells, hemoglobin, hematocrit, and white blood cells were significantly (p < .05) elevated in the PC group; however, these parameters were significantly (p < .05) improved in TG. While total antioxidant capacity and serum parameters such as total protein, albumin, and globulin were significantly (p < .05) reduced in the PC group but significantly improved (p < .05) in TG. Histological analysis revealed that smoke exposure resulted in a measurable increase in alveolar septal thickening while ethanolic extract of C. verum greatly ameliorated the histopathological changes in the lung alveoli. The gene expression analysis of EGF and EPO genes showed a significant upregulation (p < .05) of both genes in PC group while in TG, the level of both genes downregulated, in which lung damage was ameliorated due to cytoprotective effects of ethanolic extract of C. verum.


| INTRODUC TI ON
The adult lung usually remains in a dormant state during normal homeostatic phenomenon but as soon as lung tissue comes across to any sort of injury, the regenerative response is incited (Kotton & Morrisey, 2014). In lung injury, the most susceptible area of damage is lung epithelium which becomes a primary target in injury and also becomes the first one to come across with repairing process.
Lung injury either could be acute or chronic, in each case; it results in damage to the lungs which then ultimately lead to the alteration of normal epithelial homeostasis (Reiss et al., 2012). The repair mechanism of adult lungs depends upon an injury response (Crosby & Waters, 2010). Tissues, such as pancreas, lungs, and liver, exhibit a low steady-state cellular turnover although they have a responsibility to robustly replace lost or damaged cells after an injury (Hogan et al., 2014).
The role of epidermal growth factor (EGF) is reported in prenatal as well as postnatal lung development but less is known about its role in adult lung growth (Foster et al., 2002). Based upon this, the present study proposed that the EGF gene may have a potential role in regeneration of the damaged alveolar epithelium, as it is considered as a mitogenic factor having the ability to proliferate epithelial cells. Another gene selected for our proposed study is the erythropoietin gene (EPO gene); the reason why we have selected it as our gene of interest is because of its antioxidant, anti-inflammatory, effects to evaluate that either it has any relevant role in attenuating lung injury (Haine et al., 2021).
Presently, many herbal drugs are available for treating various illnesses and the list of herbal therapies is steadily growing (Huntley & Ernst, 2000) as medicinal plant extracts have fewer side effects than synthetic drugs. From the wide list of beneficial plants, Cinnamomum verum is also considered as a medicinally beneficial plant having a variety of therapeutic potentials as having an antioxidant potential, as it is rich in certain effective phyto-constituents (Faix et al., 2009).
The present study includes various parameters including gene expression analysis, oxidative stress parameters in both serum and bronchoalveolar lavage (BAL), electrocardiography (ECG) and histopathological analyses were done in order to evaluate the antioxidant potential of ethanolic extract of C. verum in ameliorating the cigarette-smoke-induced lung damage. Furthermore, our proposed study aimed to elucidate the regenerative capacities of C. verum and also to analyze the level of expression of the epidermal growth factor gene (EGF gene) and also the erythropoietin gene (EPO gene) in the alveolar epithelium, after inducing lung damage by cigarette smoke.

| Hypothesis
The EGF and EPO genes may have a potential role in regeneration of the damaged alveolar epithelium after inducing lung damage by cigarette smoke. Moreover, C. verum may have therapeutic potential against lung injury.

| Experimental animal model and experimental conditions
A total of 27 albino rats weighing 150-200 g and 6-8 weeks of age were arranged from the Animal rearing nursery of the Department of Physiology, GCUF and brought to the experimental Animal station, Department of Physiology. Consent was taken from the institutional animal ethical committee GCUF (Ref. No. GCUF/ERC/07). The experimental rats were acclimatized to the animal house with standardized environmental conditions of 25°C ± 5 temperature with appropriate humidity of 50% ± 5. The illumination was also maintained at a 12-h cycle of daylight and dark. Feed and water will be given ad libitum to rats as per their requirement during the experimental trial.
The experimental trial was composed of three groups with nine rats in each group; negative control (NC) group, positive control (PC) group, and treatment group (TG) as shown in Table 1. All were subjected to acclimatization for 1 week. For the induction of smokeinduced lung injury, the protocol was followed as described by Yu et al. (2018) with slight modification. Rats were combined in a chamber (60 × 60 × 60 cm) having two holes at the top of the chamber (outlet), lower part of the chamber (input) connected to a small air pump with a flexible hose. Lung injury was induced by exposing the rats to cigarette smoke (4 CG/day having nicotine content: 1.8 mg/cigarette) for 21 days. During the whole experimental trial of 21 days, hyperactivity, increased sweating, and whistle sound in breathing were observed. The TG (n = 9) contains rats that were orally administered with ethanolic extract of C. verum (350 mg/kg body weight) along with exposure to cigarette smoke. Another group of healthy rats was considered as the NC group (n = 9). All three groups were subdivided into three subgroups (n = 3) for the periodical sampling on the 14th, 18th, and 21st day, respectively.

| Preparation of ethanolic extract of C. verum
Cinnamon bark was procured from the herbal market and then assessed by an Expert Botanist and a voucher specimen numbered 272-bot-21 was allotted. It was kept in the herbarium of the

Body and organ weight
The weekly body weight of each rat was recorded during the experimental tenure while the weight of the lungs of each decapitated animal model was recorded on decapitation days, i.e., Days 14, 18, and 21.

Collection of blood for hematology
After cervical decapitation, blood samples were taken in an anticoagulant (ehylenediaminetetraacetic acid) tube on the specified days of decapitation to analyze the complete blood profile.

Collection of serum for oxidative stress measurements
For serum collection, blood samples were taken in clotting vials and were subjected to centrifugation at 1500 g for about 10 min. Serum was collected in Eppendorf's tubes and further analysis of oxidative stress evaluation such as total oxidant status (TOS) and total antioxidant capacity (TAC) by following the calorimetric protocols explained by Anwar et al. (2012).

| Serum analysis for total proteins and albumin
By using commercially available kits, total proteins (Bioclin® While the serum concentration of albumin is detected via colorimetric assay using Albumin Assay Kit in which a selective interaction is formed between bromocresol green and albumin as a result a chromophore is generated which is then measured spectrophotometrically at 620 nm. Final results were obtained using the following formula: Albumin g ∕ dl = ΔAbsorbance of specimen ΔAbsorbance of standard × 4

TA B L E 2 Source and names of primers (oligonucleotides)
S. no Primer Sequence Source

| Collection of bronchoalveolar lavage fluid
Bronchoalveolar lavage fluid (BALF) was performed by lavaging the lungs three to four times with normal saline which was then furtherly centrifuged for 10 min at 1500 g at 4°C (Dianat et al., 2018) and after that, the collected BALF was used to evaluate parameters such as TAC, TOS, and malondialdehyde (MDA).

| Collection of tissue sample
Histopathology Lung tissues were collected in 10% paraformaldehyde for 24 h to do fixation, and then they were dehydrated by different concentrations of ethanol. The tissues were embedded in paraffin wax thereafter and sectioned into 2-to 4μm-thin slices with the help of a microtome. The de-paraffinized sections will then be subjected to eosin and hematoxylin stains, which were further assessed under a light microscope (Huang et al., 2015).

RNA extraction
A small piece of lung tissue was taken in Trizol (Thermo Fisher Scientific) and kept at −40°C until further processing. By using Nanodrop, isolated RNA was quantified. The cDNA synthesis was done by using Revert-Aid cDNA synthesis kit (Thermo Fisher Scientific) by using equal concentrations of RNA from each sample according to the manufacturer's instructions. Finally, gene expression analyses of EGF and EPO genes were done using qRT-PCR (Biorad; BM10-QPCR96 RT 2).

| Gene expression analysis (qRT-PCR)
The gene expression analysis using primers selected for the genes (EPO and EGF genes) as shown in Table 2 was performed by using the technique of qRT-PCR.

| Phytochemical analysis of C. verum by high performance liquid chromatography
To evaluate the phytochemical constituents present in the prepared ethanolic extract of C. verum, high performance liquid TA B L E 5 Mean ± SE of body weight (g), organ weight (g), and relative organ weight (g) in negative control (NC), positive control (PC), and Cinnamomum verum treatment group (TG) at different days in cigarette-smoke-induced lung injury rat model

| Statistical analysis
Data were subjected to a two-way analysis of variance (twoway ANOVA) by using the statistical software and the level of significance between different groups was determined by applying Duncan's multiple range test in CoStat. The result was considered statistically significant at p ≤ .05. The data were represented as a mean ± standard error and the statistical evaluation was performed by using Graph Pad prism 8 and CoStat computer software. Chromatogram of ethanolic extract of C. verum is shown in Figure 1 and the relative quantity of phenolic constituents is represented in Table 4 The body weight, organ weight, and relative organ weight of rats among different groups (Table 5)  Serum albumin is considered an acute phase protein having antioxidant properties, contributes as a marker of inflammation. Thus, it implies that whenever a body is under stress condition due to any sort of injury or inflammatory response, the amount of albumin gets reduced indicating the presence of an inflammatory reaction going on in the body (Ranasinghe et al., 2013). In the present study, level of serum total protein, albumin, and globulin was significantly reduced in the PC group but significantly improved in TG (Figure 3a-c). In the present study, the level of total oxidant status and MDA was significantly higher in PC group than that of the other two groups (Figure 4a-c). However, the level of TAC was significantly lower in PC group which then seemed to be improved in the TG.

| RE SULTS AND D ISCUSS I ON
The overall mean of serum red blood cells, serum hematocrit, and serum hemoglobin level among different groups ( Table 6) were analyzed with the help of a two-way ANOVA. The data showed that there was a significant difference (p ≤ .05) among all the groups. The level of red blood cells, hematocrit, and hemoglobin was significantly higher in PC group than that in the other two groups. The level of red blood cells and hematocrit at different days, i.e., days 14, 18, and 21 was also significant (p ≤ .05). In case of cigarette-smoke-induced lung injury, the amount of carboxyhemoglobin increased which then leads to the state of hypoxia which causes excessive secretion of erythropoietin to initiate the production of RBCs (Khan et al., 2014) which further leads to an increase in hemoglobin level (Leifert, 2008). The carbon monoxide present in cigarette smoke elevates the permeability of capillaries which then results in a decrease in plasma volume.
This state mimics the state of polycythemia which is characterized by an increase in the share of RBCs in the blood volume. Thus, the hematocrit level increased due to cigarette-smoke-induced lung injury (Khan et al., 2014).
The serum white blood cells, lymphocytes, and platelets among different groups were analyzed by two-way ANOVA as shown in Table 7. The level of white blood cells and lymphocytes was TA B L E 6 Mean ± SE of red blood cell count (10 6 /μl), hematocrit (%age), and hemoglobin (g/dl) in negative control (NC), positive control (PC), and Cinnamomum verum treatment group (TG) at different days in cigarette-smoke-induced lung injury rat model TA B L E 7 Mean ± SE of white blood cell count (10 3 /μl), lymphocytes (10 3 /μl), and platelet count (10 3 /μl) in negative control (NC), positive control (PC), and Cinnamomum verum treatment group (TG) at different days in cigarette-smoke-induced lung injury rat model  (Wang et al., 2015). Furthermore, septal thickening and cellular inflammation to a greater extent were also observed in smoke-exposed rats (Gotts et al., 2017).
In the present study as shown in Figure 8, the observations obtained by electrocardiograms (ECG) obtained from PC group depicted a shortened R-R interval along with hidden P-wave and shortened QRS complex which ultimately depicted a condition of supraventricular arrhythmia which is characterized by irregular heart rate that begins above the ventricles which causes the heart to beat too fast and in irregular manner (Mutiso et al., 2014). While in the TG, this condition is quite improved with normal R-R interval and decreased heart rate as compared to that of PC group.
The mRNA expression level of EGF and EPO genes among different groups (Figure 9a,b), respectively, was analyzed with the help of two-way ANOVA. The expression level of EGF and EPO genes was significantly higher in PC group, while the expression level was downregulated in the TG. However, on different days, there was no significant difference observed in the expression level of both genes.
One study suggested that the expression capacity of epidermal growth factor receptor, i.e., EGFR is not concentrated in the case of the alveolar membrane of pathogen-free rodents like rats, mice and also there is less expression of EGFR in the upper and lower of mammalian lung airways during the normal homeostatic phenomenon. On the contrary, EGFR expression in lung airway epithelium is increased in response to any inflammatory stimuli like any sort of inflammatory mediator (TNFα), cigarette smoke, or any mechanical injury (Takeyama et al., 2001). Another study elaborated that in the case of asthma, the epidermal growth factor receptor expression is elevated in the alveolar epithelium and its activation leads to repairment in the alveolar epithelium (Burgel & Nadel, 2004). Furthermore, the hematopoietic growth factor erythropoietin (EPO) exhibits all-tissue-protective pleiotropic properties. Besides the hematopoietic effect of EPO, it has also been shown to possess certain pleiotropic properties, such as cytoprotective, anti-inflammatory, and antiapoptotic activities in the cardiovascular system, as well as in the liver and kidneys (Jelkmann, 2004).

| CON CLUS ION
The present study concluded that the cross-talk of EGF and EPO genes plays a crucial role in lung pathophysiology progression. The expression level of both EGF and EPO genes elevated with the alveolar epithelial damage in smoke-induced lung injury (PC) group in order to activate the compensatory mechanisms to attenuate the damage caused by noxious stimuli (cigarette smoke) while the level of both EGF and EPO genes downregulated in the TG (ethanolic extract of C. verum) as the HPLC analysis of C. verum showed the presence of phyto-antioxidants like flavonoids (quercetin) and phenolic constituents (P-coumaric acid) exhibiting cytoprotective, antiinflammatory, and radical scavenging properties.

ACK N OWLED G M ENTS
Authors are thanknful to all the faculty members of Department of Physiology, Government College University Faisalabad, Pakistan for providing technical support for this work.